[CANCER RESEARCH 39, 3031-3039, August 1979]
0008-5472/79/0039-0000$02.00
Isolation and Characterization of the Nuclear Matrix from Zajdela
Ascites Hepatoma Cell&
Ronald Berezney, Joseph Basler, Benjamin B. Hughes, and Steven C. Kaplan
Division of Cell and Molecular Biology, Department of Bk@4ogicalSciences, State University of New York, Buffalo, New York 14260
ABSTRACT
A procedure is reported for the isolation of Zajdela ascites
hepatoma nuclei which avoids the use of harsh detergents and
citric acid. Marker enzyme analysis, chemical composition, and
electron microscopy all indicated a high degree of purity and
intactness. The proteinaceous nucleoskeletal structure termed
the ‘
‘nuclear
matrix' ‘
was isolated from the hepatoma nuclei.
Structurally, the isolated hepatoma matrix consisted of a sur
rounding residual nuclear envelope, residual nucleoli, and an
extensive internal matrix structure. The internal matrix struc
ture, moreover, revealed a remarkable resemblance to struc
tures observed in the interchromatinic regions of intact hepa
toma nuclei. Chemically, the matrix contained 77.9% protein,
19.6% RNA, 1.0% DNA, and 1.5% phospholipid. Nearly iden
tical ultrastructure and composition is found for the corre
sponding matrices isolated from normal and regenerating liver
cells.
Although many differences were initially found in the poly
peptide profiles of hepatoma and liver matrices, isolation in the
presence of the protease inhibitors phenylmethylsulfonyl fluo
ride and sodium tetrathionate revealed that these apparent
differences were largely due to differential degradation of ma
trix polypeptides. In the presence of protease inhibitors, only
one qualitative difference was detected, a polypeptide with a
molecular weight of 100,000 unique to the hepatoma matrix.
In addition, several prominent quantitative differences were
detected. Comparison of regenerating and normal liver matrix
polypeptide profiles revealed no significant qualitative or quan
titative differences.
INTRODUCTION
Several years ago, studies were initiated (6, 7) to determine
whether the eukaryotic cell nucleus contains an overall supra
molecular structure as a basis for nuclear form and function.
Recently, this question has been answered at least partially in
the affirmative through the isolation of a similar proteinaceous
nuclear structure, termed the nuclear protein matrix, from a
variety of eukaryotic cells (8, i 0, 11, 20, 25, 27, 28, 48).
Detailed electron microscopic studies indicated that the flu
clear protein matrix represented residual nuclear protein com
ponents derived from 3 major structural regions of the in situ
nucleus (1 1): a surrounding residual nuclear envelope layer
which still contained morphologically recognizable nuclear
pore complexes; a residual nucleolar structure; and an exten
sive internal matrix which closely resembled structures ob
served in the interchromatinic matrix of intact cells. These
results are also consistent with earlier light and electron micro
scopic studies which indicated the presence of complex nu
1 This work
was supported
by USPHS
Research
Grant
Received October 2, 1978; acc€.@@@ed
April 27, 1979.
clear structures after extraction of nuclei with 2 M NaCI (24,
38, 42, 49, 50). Narayan et al. (38), for example, identified a
ribonucleoprotein network which extended from an intact flu
clear envelope to the nucleolus.
Although it is presently unknown to what degree the nuclear
matrix structure in vivo is a continuous in situ framework
structure, interchromatinic matrix structures are a character
istic feature of the in situ nucleus (4, 13, 22, 23, 29, 36, 41,
46), and observations suggest a close association between
structures of the interchromatinic matrix and the nuclear pore
complexes of the surrounding nuclear envelope (22, 36, 47).
It is conceivable, however, that the in situ interchromatinic
matrix may represent a dynamic structural system in which
macromolecular associations among various components may
be regulated (4, 11). Consistent with this possibility, isolated
nuclear matrices have the ability to reversibly expand and
contract under the influence of divalent cations (48).
Since the nuclear matrix is composed predominantly of spe
cific nonhistone matrix proteins (8, 10, 11), it is important to
consider whether distinct changes in matrix proteins provide
the molecular basis for differences in nuclear structure and/or
function observed in different cells or states of cell activity (23,
34, 41 ). Previous studies have suggested some possible dif
ferences in the molecular weights of the major matrix polypep
tides isolated from liver, HeLa, Chinese hamster ovary, and
tetrahymena
cells (1 1, 20, 25, 27, 28, 40, 48). These differ
ences, however, are based on the comparison of results from
several different studies with different matrix isolation and
SDS2:acrylamide gel electrophoretic procedures.
To provide a more definitive answer as to whether changes
in nuclear structure and function result in alterations of matrix
polypeptide composition, we have undertaken an analysis of
the polypeptide profiles of Zajdela hepatoma and normal liver
nuclear matrices isolated under identical conditions. Compari
son of the hepatoma matrix with normal liver matrix is of
particular interest since the nuclei from hepatoma cells are
characterized by irregularities in overall shape and internal
structure (35. 43).
MATERIALS AND METHODS
Zajdela ascites hepatoma cells (strain C) were transplanted
i.p. into adult male Sprague-Dawley rats (Blue Spruce Farms,
Altmont, N. V.) and were typically harvested 6 to 8 days later.
Hepatoma cells were washed 4 times in Earle's medium without
calcium or magnesium (Grand Island Biological Co., Grand
island, N. V.) containing 5 units heparin per ml with centrifu
gation at 500 rpm (50 x g) for 3 mm in a SorvaIl GLC-i
centrifuge (Dupont Instruments, Newtown, Conn.).
The hepatoma cells were swollen in TM-2 buffer at a con
GM23922.
2 Theabbreviationsused
mM
MgCl2:10
mM
Tris,
are:
pH
7.4;
SDS,
PMSF,
sodium
dodecyl
phenylmethylsulfonyl
sulfate;
TM-2
buffer,
2
fluoride.
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3031
R. Berezney et al.
centration of iO@cells/mi for 30 mm at [email protected] swollen cells
were immediately disrupted with either: (a) a Dounce homog
enizer (Clearance B, 40-mi capacity; Kontes Glass Co.) which
was calibrated in air to take —7sec for the pestle to reach the
bottom of the vessel by placing the vessel on top of the pestle
(inverted position). Seventy up-and-down strokes at O@were
judged optimal for release of intact nuclei free of adhering
contamination as monitored by phase-contrast microscopy; (b)
a tight-clearance (13 to 40 ,.tm)Potter-Elvehjem homogenizer
(Kontes Glass Co.). Forty up-and-down strokes at O@with a
speed of 1000 rpm were sufficient. Essentially equivalent re
suits were obtained with both disruption methods. The Potter
Elvehjem method was preferred because of the greater dura
bility of the Teflon pestle and the availability of homogenizers.
Hepatoma nuclei were collected by centrifugation of the
homogenate at 826 x g for 10 mm. Following one wash in TM
Nucleotidase was assayed according to the method of Aronson
and Touster (2) with 5'-AMP as substrate, and acid phospha
tase was assayed according to the method of Trouet (44) with
/3-glycerophosphate as substrate. Glucose-6-phosphatase was
assayed as previously described (12). In all cases, released P
was measured using the isobutyl alcohol -benzene extraction
procedure (30).
SDS:acrylamide gel electrophoresis was performed on 5%
acrylamide gels in 0.1 % SDS:0.05 M Tris, pH 7.4, based on a
modified procedure of Weber and Osborn (45) as detailed
earlier (1 1). Standard proteins used for molecular weight cali
bration included thyrogiobulin, fl-gaiactosidase, phosphorylase
a, bovine serum albumin, pyruvate kinase, ovalbumin, lactate
dehydrogenase, chymotrypsinogen, and myoglobin. All gels
were stained with Coomassie Blue R-250. Densitometric scans
were obtained on a Gilford Model 2520 gel scanner at 550 nm
2 buffer (826 x g, 1 0 mm), the nuclear pellet containing ‘-5 with a fixed-slit 0.05-mm plate and a scanning speed of 1 cm/
x 108 nuclei was resuspended in 30 ml of 1.8 M sucrose:TM
mm. Areas under the polypeptide peaks were quantitated by
2 bufferand centrifugedat 65,000 x umax
for 75 mm(Beckman weighing the individual peaks.
SW 25.2
rotor) through a discontinuous
sucrose gradient
con
taming 15.0 ml of 2.0 M sucrose:TM-2 buffer and 15.0 ml of
2.2 M sucrose:TM-2
buffer. Purified nuclei pelleted through the
2.2 M sucrose solution with an average recovery of 61 .9 ±
4.2%
(S.D.)
based on DNA determinations
and 55.5
± 7.2%
basedon direct
counting.
Hepatoma nuclear matrix was isolated based on procedures
reported for rat liver nuclear matrix (4, 10, 11) and involved
sequential extractions with low-magnesium (0.2 mM), high-salt
(2 M NaCI), and 1 % Triton X-1 00 solutions.
in order to prevent
gel formation during the low-magnesium and high-salt extrac
tions, the hepatoma nuclei were initially digested with pan
creatic DNase I (Worthington Biochemical Corp., Freehold, N.
J.) (5 @zg
DNase I per 108 nuclei per ml) for 15 mm at 0°.
Liver nuclei and nuclear matrix were prepared as reported
earlier (1 1). In some experiments, liver nuclei were isolated
according to the procedure described above for hepatoma
cells. Partial hepatectomies were performed on 25O-g
Sprague-Dawley rats according to the technique of Higgins
and Anderson (26). Nuclei and nuclear matrices from regen
erating liver were isolated at different periods after partial
hepatectomy in a manner identical to the procedure for normal
liver (1 1). Liver and hepatoma nuclear matrices were also
prepared in the presence of protease inhibitors by adding 1
mM PMSF
(Sigman
Chemical
Co.,
St. Louis,
Mo.)
and
1 mM
sodium tetrathionate (ICN Pharmaceuticals, Inc., Piainview, N.
V.) to all solutionsused in the matrix isolation.
Samples were prepared for thin sectioning electron micros
copy as previously described (1 1, 32, 39). All sections were
observed and photographed on a Hitachi Hu-lIC electron mi
croscope operating at 50 or 75 kV.
ANA and DNA were separated
as described
by Munro
and
Fleck (37), DNA was determined by the Burton (14) modifica
tion of the diphenylamine reaction or by direct reading at 260
nm. RNA, protein, and phospholipid were analyzed as reported
previously (12).
NADH dehydrogenase (NADH-ferricyanide reductase), suc
cinate dehydrogenase (succinate-phenazine methosulfate re
ductase), and NADH- or NADPH-cytochrome c reductases
were assayed in a Gilford Model 240 spectrophotometer
equipped with a Sargent Welch Model XKR external recorder
at a fixed temperature of 37°as described previously (12). 5'3032
RESULTS AND DISCUSSION
Purification of Zajdela Hepatoma Nuclei. Isolationof nuclei
in high purity has been difficult to achieve with rapidly growing
ascites tumor cells (15, 16, 33). As a result, many studies use
detergents or citric acid to ‘
‘clean
up' ‘
the nuclei (15, 33).
These treatments, however, result in certain modifications of
nuclear structure and extraction of nuclear components (15,
33). In order to preserve more closely the in situ structural
relationships within Zajdeia hepatoma nuclei, an isolation
method has been developed which avoids these harsh treat
ments and yields nuclei with a purity and recovery comparable
to those of liver tissue (see ‘
‘
Materials and Methods―).
The absence of detergents and/or citric acid in the nuclear
isolation method enabled us to evaluate possible cytopiasmic
contamination via marker enzyme analysis (Table 1). Appro
priate marker enzymes for plasma membranes (5'-nucieotid
Table1
Marker enzyme activities in isolated Zajdela hepatoma nuclei
compared to total homogenate
Specific activitiesare expressedas follows: 5'-nucleotidase,acid
phosphatase,and glucose-6-phosphatase,@moI
P releasedper mm
per mg protein; NADH-and NADPH-cytochromec reductase, @mol
cytochrome
c per mm per mg protein; NADH-ferricyanide
reductase,
@mol
K3Fe(CN)6
per mmper mg protein;succinatedehydrogenase,as
@zmol
2,6-dichlorophenolindophenol
per mmper mg protein.Totalac
tivity is expressed
as specific
activity
x total protein
(mg) in the
respectivefractions.
totalCell
homogenateIsolated
% of
nucleihomoge
nate activ
ity in nu
SpecificTotal ac SpecificTotal ac
activitytivityactivitytivityclei5'-Nucleotidase0.05044.40.01
10.451.0Succinate0.01816.00.0030.130.8dehydrogenaseAcid
30.541.4Glucose-6-0.06053.30.0401
phosphatase0.04540.00.01
.683.2phosphataseNADH-ferricyanide0.2241
.75.9reductaseRotenone-insensitive0.06658.60.0743.15.3NADH-cytochrome
1
990.2841
cNADPH-cytochrome
c0.01210.70.0060.252.3reductase
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39
Characterization of the Zajdela Hepatoma Nuclear Matrix
Table 2
Composition
nuclei%
of Zajdela hepatoma,
normal liver, and regenerating
liver
compositionRatiosIsolated
Protein:DNAZajdela
nuclei
hepatoma
Normal liver
Regenerating
0.15a
Protein
DNA
RNAPhospholipidRNA:DNA
71.5 ±1.Oa 20.5 ±2.2
4.4 ±1.2
72.6 ±0.5
73.7 ±1.5
3.5 ±0.2
4.3 ±0.1
20.7 ±0.6
18.3 ±1.6
± 0.3
± 0.4
3.2 ±0.4
3.49
0.17 ±0.023
± 0.25
3.51 ±0.20
3.7 ±0.20.220.24 ±0.02
4.05 ±
liver3.6
Average
± SE.
of 6 different
preparations.
ase), lysosomes (acid phosphatase), and mitochondria (succi
nate dehydrogenase) are 3- to 6-fold lower in specific activity
compared to the cell homogenate (Table 1). Total activity in
isolated hepatoma nuclei is only 0.8 to 1.4% of the total
homogenate. In contrast, glucose-6-phosphatase (3.2% of total
homogenate), NADH dehydrogenase (5.9% of total homoge
nate), and rotenone-insensitive NADH-cytochrome c reductase
(5.3% of total homogenate) are detected in much greater
amounts in hepatoma nuclei. Previous results (3, 12, 47) have
demonstrated that these characteristic enzymes of the endo
plasmic reticulum in liver cells are also endogenous compo
nents of the nuclear envelope. The typical endoplasmic retic
ulum enzyme, NADPH-cytochrome c reductase, is also de
tected in purified nuclei, although in lower amounts (2.3% of
total homogenate).
Another criterion for evaluating purity of isolated nuclei is
compositional analysis. Nuclei from specific tissues are char
acterized by distinct RNA:DNA ratios and phospholipid content.
The close similarity in the composition of hepatoma and liver
nuclei (Table 2) is thus indicative of a similar level of purity.
Note that the RNA:DNA ratio of Zajdela hepatoma nuclei (0.22)
resembles regenerating that of liver nuclei (0.24) more than
that of normal liver nuclei (0.1 7).
Purity of the isolated hepatoma nuclei was also determined
by electron microscopy. A typical survey micrograph of the
isolated Zajdela hepatoma nuclei (Fig. 1) indicates that the
nuclei are largely free of cytoplasmic contamination and that
they maintain many of the structural features characteristic of
hepatoma in situ. The bizarre shape and pleiomorphic internal
structure of nuclei and condensed chromatin patterns (Fig. 2A)
contrast sharply with the more regular arrangement of shape
and internal structure characteristic of normal liver nuclei.
Isolation and Characterization of Zajdela Hepatoma Ma
trix. Preparation of nuclear matrix from isolated hepatoma
nuclei was performed as previously described for liver nuclear
matrix (1 1). One major modification necessary for a high yield
of the hepatoma nuclear matrix was mild predigestion of the
isolation nuclei with low levels of DNase I. Study of liver nuclei
which are similarly predigested with DNase I indicated no
significant differences in morphology, composition, or poly
peptide profiles on SDS:acryiamide gels. As a rule, however,
all liver nuclear matrices reported in this study were predi
gested with DNase I under conditions identical to those for
hepatoma nuclear matrix.
The ultrastructure of the hepatoma nuclear matrix (Fig. 2B)
consists of a surrounding residual nuclear envelope, residual
nucleolar structures, and an extensive internal matrix which
extends throughout the nuclear interior. At higher magnification
(Fig. 3A), the fine structure of the hepatoma internal matrix is
shown to consist of electron-dense particles (150 to 300 A in
Table3
Composition
of Zajdela hepatoma, normal liver, and regenerating
liver
nuclear matrices
compositionProteinRNADNAPhospho
lipidZajdela
Isolated matrix%
±338
±3.3
±0.2
±0.1
hepatoma
0.3Regenerating
Normal
liver77.9
77.3 ±4.019.6 20.5 ±3.31.0 1.5 ±0.71.5 0.7 ±
±2.71
0.3a
liver77.4
9.6 ±1.91
.8 ±0.41
.2 ±
Average
± SE.of
3 to 6 different preparations.
Table4
Recovery
of macromolecules
and%
in Zajdela
regenerating
nucleiMatrix
lipidZajdela
hepatoma
Normal liver
0.3a
Regenerating
liver
Average
± SE.
hepatoma,
norma
liver,
liver matricesI
of recovery from
Protein
RNA
8.4 ±0.5a
26.2 ±3.9
0.42 ±0.1 1
4.8 ±0.9
4.6 ±1.1
22.3 ±3.2
24.7 ±1.8
1.8 ±0.2
0.38 ±0.13
0.46 ±0.082.7 2.1 ±
of 3 to 6 different
DNAPhospho
±0.3
preparations.
diameter) associated with a less electron-dense fibrous matrix.
A close resemblance of the isolated internal matrix structure to
the in situ interchromatinic matrix found between condensed
chromatin regions in intact hepatoma nuclei is apparent by
comparing Figs. 3A and 3B. A similar structural organization
for liver internal matrix was reported previously (1 1).
Table 3 demonstrates a very similar composition for hepa
toma and liver nuclear matrices (77 to 78% protein, 19 to 21%
RNA, 1 to 2% DNA, and 1 to 2% phospholipid).
Recoveries
of
total nRNA (22 to 26%), DNA (0.38 to 0.46%), and phospho
lipid (1 .8 to 2.9%) are similar (Table 4), whereas a significantly
higher percentage of total nuclear protein is recovered in
hepatoma (8.4%) compared to normal liver (4.8%) and regen
erating liver (4.6%) matrices.
Polypeptides of Zajdela Hepatoma Nuclear Matrix. The
preceding results indicate a general similarity of the hepatoma
nuclear matrix to the corresponding liver matrix with respect to
gross composition and uitrastructure. Since the matrix consists
predominantly of protein, we have investigated the hepatoma
matrix polypeptides on SDS:acrylamide gels. Densitometric
tracings of the Coomassie blue-stained gels enabled calcula
tion of polypeptide migration with a resolution of 0.1 mm or
0.1 % of the total gel length. The standard polypeptide calibra
tion curve (Chart 1) was linear in the range of 20,000 to
167,000daltons.
Over 25 polypeptides were reproducibly resolved in densi
tometric scans of 10 different hepatoma matrix preparations.
The representative scan shown in Chart 2 (Scan A) is divided
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3033
R. Berezney et al.
into 3 regions according to molecular weight: Region A,
>75,000; Region B, 42,000 to 75,000; and Region C, 15,000
to 42,000. Quantitation of the scans (Table 5) revealed the
highest amount of stained protein in Region B (47.6%) with the
remaining protein nearly equally divided between Region A
(27.3%) and Region C (25.1 %). In normal liver matrix, the
amount of stained protein in Region B (Table 5) is even more
40C
Thyroglobulin Idimer)
30C
200Thyroglobulin(monomer)
b
x
0 $-gotoctosidose
I—
I
IOC
Phosphorylosea
w
pronounced (61 .7%) with a corresponding decrease in the low
molecular-weight Region C (10.0%).
A total of 12 qualitative differences are detected in the high
molecular-weight Region A of the poiypeptide profiles. Al
though Region B contains only one polypeptide (Band 24)
which is unique to the hepatoma matrix, significant differences
are found in the relative amounts of these major matrix poly
peptides. In hepatoma matrix, the stained protein is nearly
equally divided among the 5 components (Band 28, 69,000;
Band 29, 64,000; Band 32, 55,000; Band 33, 50,000; and
Band 34, 45,000), while the liver matrix shows a predominance
of Band 28. This polypeptide (M.W. 69,000) contains over
50% of the stained protein in Region B and is by far the largest
single component in the liver matrix polypeptide profile (34.7%
of the total stained protein). A large difference is also found in
the amount of stain in the low-molecular-weight Region C
(25. 1% in hepatoma matrix and 10% in liver matrix).
Effect of Protease Inhibitors on the Polypeptide Profiles
8C
Table 5
Molecular weight distribution of matrix polypeptides isolated in the
presence or absence of PMSF:tetrathionate
determinedfrom
The percentage of Coomassie blue-stained protein was
ofthe individual weighings of the polypeptide peaks in each region
Bovine serum albumin
-J
6C
@...<@ruvOte
kinase
-J
0
\O@lbumin
4C
Lactate dehy&ogenase
either25
densitometricscan.Identicaldistributionswereobtainedwith
Valuesrepresent
or 50 /L9 of matrix
20
protein
on the SDS:acrylamide
gels.
the average of 4 separate matrix
preparations.%
Myoglobin
of total proteinmw.
stained
0
mw.mw.
i
0.2
I
0.4
I
I
i
I
0.6
0.8
MOBILITY
15,000-Matrix
I
42,000Hepatomafraction
1.0
Chart 1. Calibration curve for molelcular weight determinations of polypep
tides on SDS:acrylamide gels. The standard proteins were: thyroglobulin,
335,000 (dimer), 167,000 (monomer); $-galactosidase, 130,000; phosphorylase
a, 94,000; bovine serum albumin, 68,000; pyruvate kinase, 57,000; ovalbumin,
43,000; lactate dehydrogenase, 36,000; myoglobulin, 17,200.
25.1Liver
10.0Hepatoma
(PMSF:tetrathionate)a
Liver (PMSF:tetrathionate)a
42,000-
>75,000
75,000
27.3
29.3
44. 1
44.4
47.6
61.7
45.0
45.3
10.9
10.3
a Nuclear matrices were prepared with PMSF (1 mM) and tetrathionate (1 mM)
in all extraction solutions.
E
w
L)
z
U)
3
MOLECULAR
WEIGHT (x103)
Chart 2. Densitometric tracings of hepatoma matrix (Scan A) and liver matrix (Scan B) polypeptides resolved on SDS:acrylamide gels. The many qualitative
differences in the profiles are indicated by arrows. These differences are not apparent in nuclear matrices prepared in the presence of protease inhibitors (see text
and Table 6).
3034
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39
@
‘@
.@
g@
.@ @these
Characterization
of the Zajdela Hepatoma Nuclear Matrix
of
inreported
Hepatoma and Liver Matrices. Several investigationshavein the liver matrix profile (39.5% of total stained protein
the degradation of chromatin proteins isolated in theRegion
relativeabsence
B), but there is a nearly 50% reduction in
of protease inhibitors (17—1
9). Since the matrix pro
absenceteins
amount from 34.7% of the total stained protein in the
represent a major class of nonhistone nuclear proteins, itof PMSF:tetrathionate to 17.9% in the presence of the
proteasewas
important to determine whether the matrix proteins areinhibitors
(Table 6). In contrast, the hepatoma matrix is char
also
prone to degradation.acterized
stainQualitative
by a more equal distribution of the protein
(7.7%),toma comparison of the polypeptide profiles of hepa
among 7 polypeptides in Region B at M.W. 69,000
and liver matrices isolated in the presence of the proteaseM.W.
58,000inhibitor
64,000 (6.3%), M.W. 62,000 (5.3%), and MW.
PMSF (1 mM) (21 ) and tetrathionate (1 mM) (31 )(3.2%).
wasreveals
Note that a similar difference in protein distribution
an increase of more than 50% in the amount of stainedalso
theprotein
found in hepatoma and liver matrices isolated in
in the high-molecular-weight Region A of both hepa
inhibitors.toma
absence of protease
and liver nuclear matrices (Table 5). Moreover, the qual
Thus, use of protease inhibitors during nuclear matrix isola
itative differences denoted in Chart 2 are completely eliminatedtion
the(Table
indicates that the qualitative differences observed in
6). The only reproducible difference is a prominentpolypeptide
hepatomapolypeptide
profiles of normal liver compared to
(M.W. 100,000; Band 23) not detected in normalmatrices
degradation.liver
are largely a result of in vitro proteolytic
matrix (Table 6).Reproducible
differences, however, are still evident, suggest
Thesemolecular
Although the distribution of total stained protein in the 3ing
possible real differences in the polypeptide profiles.
weight regions of the polypeptide scans are remark
differences, moreover, are not a result of preparative proce
ably
whichbands
similar (Table 5), quantitation of individual polypeptidedures.
Nuclear matrices were prepared from liver nuclei
reveals several differences (Table 6). These include thewere
forhigh-molecular-weight
isolated by a procedure identical to that described
Band 1 (>335,000) and Band 31hepatoma
resultant(58,000),
nuclei (see ‘
‘Materials
and Methods' ‘).
The
ofconcentrated
which are, respectively, 12-fold and 3.3-fold moreliver
matrices have a polypeptide profile identical to those
isolations.(>335,000),
in the hepatoma matrix, and Bands 2matrices
prepared from the standard liver nuclei
19 (125,000), and 28 (69,000), which are 2.0- toComparison
of Normal and Regenerating Liver Matrix Poly
2.5-fold more concentrated in the liver matrix. The Band 28peptides
PMSF:Tetrathionate.polypeptide
Isolated in the Presence of
(M.W. 69,000) is still the most prominent fractionSince
the neoplastic Zajdela hepatoma cells are rapidly prolif
liverPolYpeptideprofllesofZajd:!:h:,,atornaandllvernuclear
erating in comparison to the relatively nondividing normal
Table 6
it was of interest to examine the nuclear matrix polypep
matrices
tides of rapidly proliferating normal liver cells. The polypeptide
Thepercentageof Coomassieblue-stainedproteinwasobtainedby profile of regenerating liver (24 hr after partial hepatectomy)
matrix isolated in the presence of PMSF:tetrathlonate IS vlr
weighingeach polypeptidepeak. Similarpercentageswere obtainedcells
differencesValues
identical to that of normal liver matrix. Slight
with
either 25 or 50 @g
of matrix protein on the SDS:acrylamide gels.tually
representthe averageof 4 separatematrixpreparations.in
the relative amounts of several of the polypeptides are not
% of total stained protein
statistically significant (p > 0.5) when several different prepa
rations of normal and regenerating liver matrix are examined.
.
.
Polypeptide apHepatoma Liver matrix
.
.
.
,
Identical polypeptlde profiles are obtained for
Band
A:B1
parent regenerating.
mw.
matrix (A)
(B)
Ratioa
partial2 >335,000
matrix isolated 3, 6, 12, 48, and 72 hr after
1 .2
0.1
12liver
>335,000
0.1
0.2
0.6
0.5
>335,000
0.8
0.8
270:000
200,000
1 .2
1 .9
1 .3
1 .7
1 55,000
145,000
1 .3
2.4
1 .5
2.3
130,000
125,000
115,000
0.9@g@@ggg
3.3
2.4
3.2
3.4
5.8
3.6
Moreover,25
24
95:000
3.2
3.8
close similarity in the matrix polypeptide profiles isolated
from normal and regenerating liver cells suggests that a
1.0
the liver cell from a nonproliferating to a proliferating state is
not correlated with significant alterations in the protein com
position of the respective nuclear matrices. Therefore, differ
0.9in
1 .1
in the polypeptide profiles of hepatoma and normal liver
matrices appear to be specific for the hepatoma cell
0.9than
a result of a general difference between proliferating
1.0
cells. Whether these differences are in some
1.0
.
.
manner a reflection
of the neopiastlc
state ,, of the hepatoma
0.4nonproliferating
cells or are primarily a result of special properties of
tumor cells is not clear. Studies of nuclear matrices
0.8ascites isolated from solid tumors should resolve this issue.
88,000
3.5
3.7
0.9
change6
5
>335:000
9
12
@
rather1
5
and16
18
19
these21
@:@gg
in29
more30
@
0.5hepatectomy.@g
@;
cells33
@.; 3.7
0.7
differences
are based
on one-dimensional
eiectropho
64,000
6.3
6.0
62,000
5.3
4.5
50,000
in4@@gg@
7.9
5.4
1.5it)
@:@gg10:3
2.5
3.0
17,500
differences
?:?ences
retic separation according to molecular weight. Studies
with 2-dimensional gel systems should provide
1.1progress
1.2
information.
Zajdela hepatoma cells (Figs. 1 and 2A) and neoplastic
consequence38
37
29:000
a Major
1 .2The
between
hepatoma
2.5
3.0
and liver matrices
@precise
1.0the
1.0of
are in italics.
general
(1 3, 35, 43) are characterized
by abnormalities
shape, size, and internal structure of the cell nucleus. At
least some of these structural lesions may be a
distinct changes in the nuclear matrix of neoplastic cells.
Further studies of matrix proteins from normal and neoplastic
AUGUST1979
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3035
R. Berezney et al.
cells may, therefore, yield important clues for resolving the
molecular basis for these alterations in nuclear structure.
Although it is not clear to what extent these structural ab
normalities are related to changes in nuclear functioning, it is
important to realize that an actual association of nuclear func
tioning with the nuclear matrix structure is suggested by the
presence of newly replicated DNA (4, 9, 10, 47) and the active
phosphorylation of high-molecular-weight matrix polypeptides
(1 , 4, 5, 10). Whether or not matrix proteins have specific roles
in this functioning or its regulation remains to be determined.
ACKNOWLEDGMENTS
We wish to thank Dr. Edward J. Sarcione of the Roswell Park Memorial
Institute for the Zajdela ascites hepatoma cells.
REFERENCES
1. Allen, S. L., Berezney, R., and Coffey, D. S. Phosphorylation of nuclear
matrix proteins during rat liver regeneration. Biochem. Biophys. Res. Com
mun., 75:111—116,1977.
2. Aronson, N. N., and Touster, 0. Isolation of rat liver plasma membrane
fragments in isotonic sucrose. Methods Enzymol., 31: 90—102,1974.
3. Berezney, R. Large scale isolation of nuclear membranes from bovine liver.
Methods Cell Biol., 8: 205-228, 1974.
4. Berezney, R. Dynamic properties of the nuclear matrix. In: H. Busch (ed),
5.
6.
7.
8.
9.
10.
11.
12.
13.
The Cell Nucleus, Vol. 7, pp. 413—456.New York: Academic Press, Inc.,
1979.
Berezney, Allen, S., and Coffey, D. S. Phosphorylation of the nuclear protein
matrix. J. Cell Biol., 70 (2. Part 2): 305a, 1976.
Berezney, R., and Coffey, D. S. Isolation of a nuclear structural complex
from mammalian nuclei. J. Cell Biol., 59 (2, Part 2): 22a, 1973.
Berezney, R., and Coffey, D. S. Identification of a nuclear structural protein
network in rat liver nuclei. Fed. Proc., 33: 1395, 1974.
Berezney, R., and Coffey, D. S. Identification of a Nuclear Protein Matrix.
Biochem. Biophys. Res. Commun., 60: 14 10- 14 17, 1974.
Berezney, R., and Coffey, D. S. Nuclear protein matrix: association with
newly synthesized DNA. Science, 189: 291 —293.1975.
Berezney, R., and Coffey, D. S. The nuclear protein matrix: isolation,
structure and functions. Adv. Enzyme Regul., 14: 63—1
00, 1976.
Berezney, R., and Coffey, D. S. Nuclear matrix. Isolation and characterization
of a framework structure from rat liver nuclei. J. Cell Biol., 73: 616—637,
1977.
Berezney, R., Macaulay, L. K., and Crane, F. L. The purification and
biochemical characterization of bovine liver nuclear membranes. J. Biol.
Chem., 247: 5549-5561, 1972.
Bernard, W., and Granbonlan, N. The fine structure of the cancer cell
nucleus. Exp. Call Res. Suppl., 9: 19—53,1963.
i 4. Burton, K. Determination of the DNA concentration with diphenylamine.
15.
16.
17.
18.
19.
Methods enzymol., 12B: 163—1
66, 1968.
Busch, H., and Daskel, Y. Methods for isolation of nuclei and nucleoli.
Methods Cell Blol., 16: 1—43,1977.
Busch, H., Hodnett, J. L., Morris, H. P., Neogy, R., and Unuma, T. Isolation
of nuclei and nucleoli of Morris hepatoma cells. Methods Cancer Res., 4:
179-190, 1968.
Carter, D. B., and Choe, C. B. Chromatin-bound protease: degradation of
chromosomal proteins under chromatin dissociation conditions. Biochemis
try, 15: 180-185, 1976.
Choe. C. B., and Carter, D. B. Degradation of chromosomal proteins during
dissociation and reconstitution of chromatin. Biochem. Biophys. Res. Com
mun., 57: 740—746,1974.
Chong, M. T., Garrard, W. T., and Bonner, J. Purification and properties of
a neutral protease from rat liver chromatin. Biochemistry, 13: 5128—5134,
1974.
20. Comings, D. E., and Okada, T. A. Nuclear proteins Ill. The fibrillar nature of
the nucleus matrix. Exp. Cell Res., 103: 341—360,1976.
21 . Fahnney, D. E., and Gold, A. M. Sulfonyl fluorides as inhibitors of esterases
I. Rates of reaction with acetylcholinesterase, a-chymotrypsin and trypsin.
J. Am. Chem. Soc.. 85: 997-1000, 1963.
3036
22. Franke, W. W., and Falk, H. Appearance of nuclear pore complexes after
Bernhard's staining procedure. Histochemie, 24: 266—278,1970.
23. Frenster, J. H. Ultrastructure and function of heterochromatin and euchro
maim. In: H. Busch (ad.), The Cell Nucleus, Vol. 1, pp. 565—580.New York:
Academic Press, Inc., 1974.
24. Georgiev, G. P., and Chentsov, S. The structural organization of nucleo
chromosomal ribonucleoproteins. Exp. Cell Res.. 2 7: 570—572,1962.
25. Herlan, G., and Wunderlich, F. Isolation of a nuclear protein matrix from
Tetrahymena macronuclei. Cytobiologie, 13: 291 -296, 1976.
26. Higgins, G. M., and Anderson, R. M. Experimental pathology of the liver I.
Restoration of the liver of the white rat following partial surgical removal.
Arch. Pathol., 12: 186—202.1931.
27. Hildebrand, C. E., Okinaka, R. T., and Gurley, L R. Existence of a residual
nuclear protein matrix in cultured Chinese hamster cells. J. Cell Biol., 67:
169a, 1975.
28. Hodge, L. D., Mancini, P., Davis, F. M., and Heywood, P. Nuclear matrix of
HeLa S3 cells. Polypeptide composition during adenovirus infection and in
phases of the cell cycle. J. Cell Biol., 72: 194—208,1977.
29. Kubai, D. F., and Ris, H. Division in the Dinoflagellate Cypodinium cohnii. J.
Cell Biol., 40: 508-528, 1969.
30. Lindberg. 0., and Ernster, L. Determination of organic phosphorous com
pounds by phosphate analysis. Methods Biochem. Anal., 3: 1—22,1955.
31 . Liu, T. Y. Demonstration
of the presence
of a histidine
residue
at the active
site of a streptococcal proteinase. J. Biol. Chem., 242: 4029—4032,1967.
32. Luft, J. H. improvements in epoxy resin embedding methods. J. Biophys.
Biochem. Cytol., 9: 409-41 4, 1961.
33. Magliozzi, J., Pino, D.. Lin, C., Ortman, R., and Dounce, A. Ratios of nuclear
proteins to DNA for rat and mouse tumors and possible effect of cytoplasmic
fibrils in isolating nuclei. Exp. Cell Res., 67: 111—1
23, 1971.
34. Maul, G. G., Maul, H. M., Scogna, J., Liberman, M. W., Stein, G. S., Hsu, B.
Y. I., and Borun, I. W. Time sequence of nuclear pore formation in phyto
hemagglutinin-stimulated lymphocytes and in HeLa cells during the cell
cycle. J. Cell Biol., 55: 433—447,1972.
35.
36.
37.
38.
39.
40.
Miyagi,
K., and Steiner,
J. W. Fine structure
of interphase
liver cell nuclei in
subacute ethionine intoxication. Exp. Mol. Pathol., 4: 525—566,1965.
Monneron, A., and Bernhard, W. Fine structural organization of the inter
phase nucleus in some mammalian cells. J. Ultrastruct Res., 27: 266—288,
1969.
Munro, H. N., and Fleck, A. The determination of nucleic acids. Methods
Biochem. Anal., 14: 113-1 76, 196g.
Narayan, K. S., Steele, W. J., Smetana, K., and Busch, H. Ultrastructural
aspects of the ribonucleoprotein network in nuclei of Walker tumor and rat
livers. Exp. Cell Res., 46: 65—77,1967.
Reynolds, E. S. The use of lead citrate at high pH as an electron-opaque
stain in electron microscopy. J. Cell Biol., 17: 208—21
2, 1963.
Riley, D. E., Keller, J. M., and Byers, B. The isolation and characterization
of nuclear ghosts from cultured HeLa cells. Biochemistry, 14: 3005—3013,
1975.
41 . Simard, R., Langelier, Y., Mandeville, R., Maestracci, N., and Royal, A.
Inhibitors as tools in elucidating the structure and function of the nucleus.
In: H. Busch (ed), The Cell Nucleus, vol. 3, pp. 447—487.New York:
Academic Press, Inc., 1974.
42. Smetana, K., Steele, W. J., and Busch, H. A nuclear ribonucleoprotein
network. Exp. Cell Res., 31: 198—201
, 1963.
43. Svoboda, D., and Higginson, J. A comparison of ultrastructural changes in
rat liver due to chemical carcinogens. Cancer Res., 28: 1703—1
733, 1968.
44. Trouet, A. Isolation of modified liver lysosomes. Methods Enzymol., 31:
323-329, 1974.
45. Weber, K., and Osborn, M. The reliability of molecular weight determinations
by dodecyl
sulfate-polyacrylamide
gel electrophoresis.
J. Biol. Chem.,
244:
4406—4412,1969.
46. Wolfe, S. Biology of the Cell, Ed. 1•
pp. 184- 186. Belmont, Calif.: Wadsworth
Publishing Co., 1972.
47. Wunderlich, F., Berezney, A., and Kleinlg, H. The nuclear envelope: an
interdisciplinary analysis of its morphology, composition and functions. In:
D. Chapman and D. F. H. Wallach (eds.), Biological Membranes, Vol. 3, pp.
241 -333. New York: Academic Press, Inc., 1976.
48. Wunderlich, F., and Herlan, G. A reversibly contractile nuclear matrix. Its
isolation, structure and composition. J. Cell Biol., 73: 271 —278,1977.
49. Zbarsky, I. B., Dmitrieva, N. P., and Yermolayeva, L. P. On the structure of
tumor cell nuclei.
Exp. Cell Res., 2 7: 573—576,
1962.
50. Zbarsky, I. B., and Georgiev, F. P. Cytological characteristics of protein and
nucleoprotein fractions of cell nuclei. Biochim. Biophys. Acta, 32: 301—302,
1959.
CANCERRESEARCHVOL. 39
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Characterization
of the Zajdela Hepatoma Nuclear Matrix
A
C:
‘,‘@:
‘p
Fig. 1 . Survey
electron
micrograph
of isolated
Zajdela
hepatoma
nuclei.
The level of visible cytoplasmic
contamination
was very low (small
arrow).
Over 90%
of
the nuclei were judged intact with large regions of the outer nuclear membrane visible. Occasional disrupted nuclei were observed (large arrow). Note the irregularities
in nuclear shape. x 9,200; bar, 4 yam.
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3037
R. Berezney et al.
h-@@@7@)
‘.
@
@
_______
Fig. 2. A, a typical isolated
hepatoma
nucleus.
The outer-nuclear
j@vti
membrane
2B
is clearly visible. Note the irregularities
Iin shape and pattern
I
of condensed
chromatin.
NE,nuclearenvelope;N, nucleolus;IM, interchromatinic
matrix;CC,condensedchromatin.x 16,000;bar, 1 yam.B, isolatedhepatomamatrix.RE,residualnuclear
envelope; N, residual nucleolus; IM, internal matrix. x 25,000; bar, 1 yam.
3038
CANCERRESEARCHVOL. 39
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Characterization
of the Zajdela Hepatoma Nuclear Matrix
Fig. 3. ultrastructural comparison of the internal matrix region of an isolated hepatoma nuclear matrix with the in situ matrix of a hepatoma cell. Both structures
consist of dense granules enmeshed in a fibrous matrix. (Compare regions enclosed by dashed lines.) A. internal matrix of isolated hepatoma matrix. x 105,000; bar,
0.2 yam. B. nuclear
interior
of a hepatoma
cell. N. nucleolus;
CC, condensed
chromatin;
IM. interchromatinic
matrix.
x 105.000;
bar, 0.2 yam.
AUGUST1979
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3039
Isolation and Characterization of the Nuclear Matrix from
Zajdela Ascites Hepatoma Cells
Ronald Berezney, Joseph Basler, Benjamin B. Hughes, et al.
Cancer Res 1979;39:3031-3039.
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